(J-382) L-DNA Sensing of Annealing as Cycling Control Mitigates Unextracted Nasopharyngeal PCR Interferents

Introduction:: PCR DNA extraction is often used to overcome interference in patient samples. This step, however, introduces additional cost and complexity into PCR testing. Recent reports suggest that some unextracted samples can be used as a screening tool for infection, like SARS-CoV-2. In the N2 region of the original CDC assay, however, unextracted samples have delayed C­_t values and false negative results that could be due to interferents. Previous research demonstrated that changing the annealing temperature from the value prescribed for the N2 reaction in the CDC procedure can “rescue” unextracted samples and produce results that approach those of extracted samples, in both nasopharyngeal matrix and NaCl concentrations similar to those of common viral transport media (VTM)¹. Moreover, we have previously presented a novel “adaptive” PCR instrument that uses primer-matched, fluorescently end-labeled L-DNA probes to monitor primer annealing as it occurs. By using these probes to make cycling decisions, we have demonstrated that we can adapt to changing sample compositions. Therefore, we sought to use the SARS-CoV-2 N2 sequence as an exemplar for potential use in other potentially interferent-sensitive reactions. In this work, we demonstrate that using our L-DNA probes to control thermal cycling automatically sets the annealing switch point, and therefore allows calibration-free PCR in unextracted nasal samples.

Materials and Methods:: All reactions were carried out using the TaqPath 1-Step RT-qPCR kit, in a 20 µL sample volume. All primers were added at 500 nM, with probes at 150 nM. To emulate unextracted nasopharyngeal samples, 5 µL of heat-inactivated nasopharyngeal matrix replaced the equivalent amount of water, while 5 µL of 140 mM NaCl was used for the simulated VTM samples to reach a final NaCl concentration of 35 mM. The control samples were performed in a molecular grade water background. To simulate positive samples, 10⁶ copies of synthetic N2 SARS-CoV-2 were spiked into each with 10⁶ copies of synthetic N1 RNA multiplexed to serve as a positive control. An L-DNA mix of a high melt temperature sequence for melt control, N2 L-DNA, and an L-DNA beacon to control RT temperature were added to each sample in total concentrations matching that of our N2 primer-probe mix. Water, nasopharyngeal, and VTM simulant samples were placed in the adaptive PCR instrument and cycled according to the CDC EUA procedure: a 15 minute, 50°C RT step followed by 45 PCR cycles, with the L-DNA fluorescent probes used to set the cycling behavior of the instrument rather than any thermal measurements. C_t values were calculated with LinReg PCR for each sample. C_t difference from water was compared to zero with the student’s t-test. Each sample group consisted of 6 samples in experimental background with 6 water samples for comparison to control for potential error across sample preparation and evaluate performance against “ideal” samples.

Results, Conclusions, and Discussions::

All 24 samples were positive for both the positive N1 control and the N2 test sequence, with no false negatives seen across all replicates in all groups. The change in C_t values versus water as well as a 95% confidence interval for their calculations are shown in Figure 1. Average C_t for the N1 sequences in nasopharyngeal background was 27.3, while the average for simulated VTM was 27.6. In comparison, the N1 C_t values were 26.8 and 27.2 for the comparative water sample groups for nasopharyngeal and 35 mM NaCl, respectively. Neither change in C_t versus water was statistically significant, but that was expected for the N1 control. Conversely, N2 nasopharyngeal and simulated VTM samples had C_t values of 21.9 and 21.3 respectively, compared to 21.2 and 19.9 for their water control groups. The N2 sequence amplified in all cases, overcoming the failures seen previously using the CDC procedure¹. Moreover, the C_t did not differ significantly from the water control samples, indicating that the adaptive PCR process restores the N2 reaction in sample backgrounds that would ordinarily interfere with its amplification. Overall, our adaptive instrument’s performance with the N2 sequence provides promising evidence that it can “rescue” these sensitive reactions from failure due to common interferents found in unextracted samples.

Acknowledgements (Optional): : We would like to thank Jonathan Schmitz at Vanderbilt Medical Center for nasopharyngeal background samples.

References (Optional): : (1)  Victoriano, C. M.; Pask, M. E.; Malofsky, N. A.; Seegmiller, A.; Simmons, S.; Schmitz, J. E.; Haselton, F. R.; Adams, N. M. Direct PCR with the CDC 2019 SARS-CoV-2 Assay: Optimization for Limited-Resource Settings. Sci Rep 2022, 12 (1), 11756. https://doi.org/10.1038/s41598-022-15356-7.